CN109134615B - Preparation method of bivalirudin - Google Patents
Preparation method of bivalirudin Download PDFInfo
- Publication number
- CN109134615B CN109134615B CN201811101059.4A CN201811101059A CN109134615B CN 109134615 B CN109134615 B CN 109134615B CN 201811101059 A CN201811101059 A CN 201811101059A CN 109134615 B CN109134615 B CN 109134615B
- Authority
- CN
- China
- Prior art keywords
- gly
- fmoc
- bivalirudin
- pro
- resin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to the field of polypeptide synthesis, and particularly relates to a bivalirudin preparation method. The invention adopts amino acid fragments without side chain protection between Fmoc-Pro-Gly-Gly-OH and Fmoc-Gly-Gly-OH as raw materials, has simple process, controllable quality and easy industrial production, better avoids the difficulties of poor solubility, difficult recrystallization and the like of Fmoc-Pro-Gly-Gly-Gly-Gly-OH and the like through the Fmoc-Pro-Gly-Gly-OH, and simultaneously avoids the generation of impurities of Bivalirudin +/-1 Gly and Bivalirudin +/-2 Gly. The solid-phase synthesis bivalirudin is simple to operate and easy for industrial production, TFA is adopted for cracking, HF is avoided, the safety is high, the purity of the synthesized bivalirudin crude product is more than 93%, and the yield is more than 99%. Ammonium acetate solution and acetonitrile are adopted for one-step purification, TFA/water and acetonitrile are used for two-step purification, and the refined peptide obtained by freeze-drying has the purity of over 99.8 percent and the yield of over 58 percent, and the purification process is simple and is easier for industrial large-scale production.
Description
Technical Field
The invention relates to the field of preparation of polypeptide bulk drugs, and in particular relates to a bivalirudin preparation method.
Background
Bivalirudin, the English common name of Bivalirudin and the trade name of Angiomax, is a linear twenty-peptide, and the molecular structural formula is as follows:
the amino acid sequence is as follows:
D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ilu-Pro-Glu-G lu-Tyr-Leu
the molecular formula is as follows: c98H138N24O33Molecular weight: 2180.32, CAS: 128270-60-0.
Bivalirudin (trade name: Angiomax) for injection is a polypeptide product meeting the requirements of the FDA, is a direct, specific and reversible thrombin inhibitor and is used as an anticoagulant for adult time-selective Percutaneous Coronary Intervention (PCI). Bivalirudin, available from The pharmaceutical Company of The United states (The medicinal Company). FDA approval was given in the year 2000 at 12 months.
Bivalirudin is a direct thrombin inhibitor and has an inhibiting effect by specifically binding to the catalytic site and the anionic external binding site of thrombin on free thrombin and thrombi. Thrombin, a serine protease, plays an important role in the process of thrombus formation by first decomposing fibrinogen into fibrin monomers and then activating thrombin factor XIII to XIIIa to covalently link fibrin into a stable network, thereby forming a thrombus. The thrombin can also activate thrombin factors V and VIII to further promote the formation of thrombin, and can also activate platelets to cause platelet aggregation and release platelet aggregates. The binding process of bivalirudin and thrombin is reversible, and the thrombin can restore the original biological activity by slowly hydrolyzing peptide bonds between bivalirudin Arg3-Pro 4. In vitro studies have shown that bivalirudin not only inhibits free thrombin, but also inhibits thrombin bound to blood clots without being neutralized by substances released from platelets, and it prolongs the normal human plasma activated partial thromboplastin time (aPTT), Thrombin Time (TT) and Prothrombin Time (PT) in a linear relationship with the concentration of bivalirudin, but it is unclear whether such a correlation exists in clinical applications.
The Bivalirudin structure contains a-Gly-Gly-Gly-Gly-Gly-segment, impurities of Bivalirudin +/-1 Gly and Bivalirudin +/-2 Gly are easily generated before and after a main peak due to the characteristics of Gly in the solid-phase stepwise synthesis process, and are difficult to remove in subsequent separation, the Fmoc-Gly-Gly-Gly-OH tetrapeptide segment is synthesized under the liquid phase condition by patent CN102260323A, then the Fmoc-Gly-Gly-Gly-Gly-Gly-OH tetrapeptide segment is connected onto peptide resin, and the residual amino acids are connected step by step, but the Fmoc-Gly-Gly-Gly-OH tetrapeptide segment has poor solubility and extremely difficult recrystallization, and the high-purity Fmoc-Gly-Gly-Gly-Gly-OH tetrapeptide segment is difficult to obtain. Bivalirudin +/-1 Gly, Bivalirudin +/-2 Gly and Bivalirudin-Arg impurities are reduced by using Fmoc-Gly-Gly-Gly-OH, fragments Fmoc-Arg (Pbf) -Pro-OH and Fmoc-Gly-Gly-Gly-Asn (R) -Gly-Asp (OtBu) -OH as raw materials for synthesizing fragments of Bivalirudin in patents CN102286076A and CN102532274A of Chengdou biopharmaceutical Limited company, but the multi-fragment synthesis process is complex, poor in quality controllability and not easy for industrial production. Patent CN102731624A synthesizes Fmoc-D-Phe-Pro-Arg (Pbf) -Pro-Gly-Gly-Gly-OH by using acid-unstable CTC resin, then Gly-Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu) -Ile-Pro-Glu (OtBu) -Glu (Ot Bu) -Tyr (tBu) -Leu-Wang Resins are received under the condition of twice feeding, and then TFA component is cracked to obtain target peptide. Qilu pharmaceutical limited patent CN103242431A adopts Fmoc-Pro-OH and H-Gly-Gly-Gly-Gly-OH in liquid phase to synthesize Fmoc-Pro-Gly-Gly-Gly-Gly-OH pentapeptide fragments as raw materials, and Fmoc-Leu-Wang resin as a solid phase carrier to synthesize Bivalirudin, thereby effectively avoiding the generation of impurities of Bivalirudin +/-1 Gly and Bivalirudin +/-2 Gly. Although the solubility of Fmoc-Pro-Gly-Gly-Gly-Gly-OH is better than that of Fmoc-Gly-Gly-Gly-OH, the preparation of the pentapeptide fragment Fmoc-Pro-Gly-Gly-Gly-Gly-OH still cannot obtain a fragment pentapeptide product by extraction, but the fragment pentapeptide product is obtained from water, so the industrial production is not easy, on the other hand, the recrystallization is difficult, the impurities of Fmoc-Pro-Gly-Gly-Gly-Gly-Gly-OH, Fmoc-Pro-Gly-Gly-OH and Fmoc-Pro-Gly-Gly-OH still exist, and the recrystallization quality controllability is poor after the impurities are dissolved by DMF. In patent 201510754607.3, fragments Pro-Gly and Gly-asn (trt) are used as raw materials for solid phase synthesis, wherein the fragments Gly-asn (trt) have side chain protecting groups, and both liquid phase acid regulation and solid phase cracking can cause the dropping of the protecting groups, resulting in poor quality controllability and inconvenience for large-scale production. In patent 201510005428.X, Fmoc-Gly-Gly-OH is used as a solid phase synthesis raw material to carry out Bivalirudin solid phase synthesis, but researches show that free H-Gly-OH and H-Gly-Gly-OH amino acids exist in the raw material Fmoc-Gly-Gly-OH, and the risk of generating Bivalirudin +/-1 Gly and Bivalirudin +/-2 Gly impurities is increased. In view of the above, a bivalirudin custom preparation method which is easy to be industrially produced and has controllable quality is needed.
The invention content is as follows:
aiming at the defects of the prior art, the invention summarizes the preparation method of Bivalirudin, and invents the preparation method of Bivalirudin +/-1 Gly and Bivalirudin +/-2 Gly, which not only can effectively avoid the generation of impurities of the Bivalirudin +/-1 Gly, but also has the advantages of easily controlled quality and easy industrialization.
The technical scheme of the invention is as follows:
a method for preparing bivalirudin comprises the following steps:
(1) adopting Fmoc-Leu-Wang resin or Fmoc-Leu-CTC resin as a solid phase carrier, adding a deprotection agent, and removing an Fmoc protecting group on the resin;
(2) sequentially coupling the resin with the Fmoc protecting group removed in the step (1) with Fmoc protecting amino acids according to the sequence of bivalirudin in the presence of an activating agent to obtain bivalirudin peptide resin with a protected side chain:
R1-D-Phe-Pro-Arg (Pbf) -X-Y-Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu) -Ile-Pro-Glu (OtBu) -Tyr (tBu) -Leu-resin
Wherein R is1Is Fmoc or Boc;
x is Pro-Gly-Gly;
y is Gly-Gly;
(3) cracking the bivalirudin peptide resin with the side chain protected obtained in the step (2), and settling to obtain a bivalirudin crude product;
(4) and (4) purifying and freeze-drying the bivalirudin crude product in the step (3) to obtain a bivalirudin fine product.
The substitution range of the Fmoc-Leu-Wang resin or the Fmoc-Leu-CTC resin in the step (1) is 0.40-0.80 mmol/g, preferably 0.6-0.7 mmol/g;
in the step (1), the deprotection agent is 20% v/v piperidine/N, N-Dimethylformamide (DMF) solution, the mass volume ratio of the resin to the deprotection agent is 1: 6-12, the deprotection reaction time is 5-20 min, preferably, the dosage of the deprotection agent is 8-10 times of the mass of the resin, and the deprotection reaction time is 8-12 min.
The Fmoc protected amino acid form in the step (2) is as follows:
Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-Gly-Gly-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-D-Phe-OH or Boc-D-Phe-OH, the multiple of the charge being preferably 2.0 to 3.0 times the scale of synthesis;
the activating agent in the step (2) is one of HOBT/DIC, HOAT/DIC, HBTU/HOBT/DIEA and HATU/HOAT/DIEA, and preferably, the feeding amount of the activating agent is 2.2-3.3 times that of the resin; the coupling time is 1-5 h, preferably 2-3 h.
The Fmoc-Pro-Gly-Gly-OH fragment synthesis steps are as follows: by adopting a liquid phase synthesis method, taking H-Gly-Gly-OH, Fmoc-Pro-Osu and sodium carbonate as raw materials, taking 1, 4-dioxane and water as solvents, carrying out TLC detection reaction, adding ethyl acetate which is 5-8 times of the feeding times of Fmoc-Pro-Osu, extracting, concentrating, crystallizing, recrystallizing, filtering and drying to obtain Fmoc-Pro-Gly-Gly-OH.
The molar ratio of the H-Gly-Gly-OH to the sodium carbonate is preferably 1: 0.8-1.0, and the molar ratio of the Fmoc-Pro-OSu to the diglycine is preferably 1: 1.2-1.4.
In the step (3), the cracking solution used for cracking is one or more of 90-95% TFA and phenylmethyl sulfide, ethanedithiol, phenol, water and triisopropyl silane, and preferably TFA/H2O/TIS/phenylmethyl sulfide; the amount of the lysis solution is preferably 6-8 times of the mass of the peptide resin; the cracking reaction time is preferably 2.0-3.0 h.
In the step (4), the purification adopts a reversed phase C18 or C8 preparation column, and the mobile phase A is as follows: performing gradient elution on 50-100 mmol/L ammonium acetate solution and B-phase acetonitrile, and performing one-step purification to obtain a pure qualified solution with the purity of more than 96%; and then, performing gradient elution by adopting mobile phase A phase 0.01-0.05% TFA/water and phase B acetonitrile at the flow rate of 600ml/min to complete salt conversion purification (two-step purification), obtaining two pure qualified solutions, concentrating the qualified solutions, and freeze-drying to finally obtain a refined peptide sample with the purity of more than 99.5%.
The invention has the beneficial effects that:
1. the invention adopts amino acid fragments without side chain protection between Fmoc-Pro-Gly-Gly-OH and Fmoc-Gly-Gly-OH as raw materials, has simple process, controllable quality and easy industrial production, synthesizes Fmoc-Pro-Gly-Gly-OH by Fmoc-Pro-Osu and H-Gly-Gly-OH raw materials in a liquid phase manner, has the purity of more than 99.9 percent and the yield of more than 92 percent through recrystallization, better avoids the difficulties of poor solubility, difficult recrystallization and the like of the Fmoc-Pro-Gly-Gly-Gly-OH and the like through the Fmoc-Pro-Gly-Gly-OH, and simultaneously avoids the generation of impurities of Bivalidudin +/-1 Gly and Bivaludirin +/-2 Gly.
2. The solid-phase synthesis of bivalirudin is simple to operate and easy for industrial production, and the TFA is adopted for cracking, so that the use of HF is avoided, and the safety is high; meanwhile, Fmoc-Pro-Gly-Gly-OH and Fmoc-Gly-Gly-OH are used as raw materials, the risk of generating impurities by using single Fmoc-Gly-Gly-OH as the raw materials is reduced, the generation of impurities of Bivalerudin +/-1 Gly and Bivalerudin +/-2 Gly is effectively avoided, the purity of the Bivalirudin crude product is more than 95%, and the yield is more than 99%.
3. The purification steps of the invention adopt ammonium acetate solution and acetonitrile for one-step purification, TFA/water and acetonitrile complete two-step purification, and the refined peptide obtained by freeze-drying has the purity of more than 99.8 percent and the yield of more than 59 percent, and the purification process is simple and is easier for industrialized mass production.
The specific implementation mode is as follows:
the present invention is further described in the following detailed description, which is for the purpose of illustration only, and the scope of the invention is not limited to these examples, and it will be understood by those skilled in the art that various equivalent substitutions and modifications may be made within the scope of the invention.
The raw materials and reagents mentioned in the specification have the meanings as follows:
CTC resin 2-chlorotrityl chloride resin
Wang Resins resin
Fmoc 9-fluorenylmethyloxycarbonyl
tBu tert-butyl
Boc tert-butyloxycarbonyl group
Pbf 2,2,4,6, 7-pentamethylbenzofuran-5-sulfonyl
Trt trityl radical
DCM dichloromethane
DMF N, N-dimethylformamide
DMAP 4-dimethylaminopyridine
DIPEA N, N-diisopropylethylamine
DIC N, N-diisopropylcarbodiimide
HBTU benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
HATU 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
TBTU O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate
HOBT 1-hydroxybenzotriazole
HOAT 1-hydroxy-7-azobenzotriazol
DIEA N, N-diisopropylethylamine
TFA trifluoroacetic acid
TIS Triisopropylsilane
HOSu N-hydroxysuccinimide
Ninhydrin as Kaiser test detection reagent
Example 1: synthesis of Fmoc-Pro-Gly-Gly-OH
Weighing Fmoc-Pro-OSu (43.45g,100mmol), adding 200ml of solvent 1, 4-dioxane for dissolution, weighing H-Gly-Gly-OH (18.48g,140mmol) and sodium carbonate (20.14g,190mmol), adding 200ml of water for dissolution, slowly adding Fmoc-Pro-OSu solution, detecting reaction by TLC (thin layer chromatography), reacting at room temperature for 3.0 hours, concentrating under reduced pressure to remove 1, 4-dioxane, adding 300ml of ethyl acetate, adjusting pH to 2-3 with 2N diluted hydrochloric acid, extracting and retaining an ethyl acetate phase, concentrating and crystallizing, filtering, drying, adding 360ml of ethyl acetate for redissolving, continuously recrystallizing with ethyl acetate, concentrating, freezing and crystallizing, performing suction filtration and drying to obtain Fmoc-Pro-Gly-OH, weighing 41.84g, obtaining 92.77% of total yield and 99.98% of purity.
Example 2: synthesis of bivalirudin peptide resin with fully protected side chain
Weighing 25.0g (substitution degree of 0.6mmol/g) of Fmoc-Leu-Wang resin with the synthetic mole number of 15.0mmol, adding the Fmoc-Leu-Wang resin into a polypeptide synthesis reactor, adding 250.0ml of DMF to swell the resin for 30min, then pumping off the DMF, carrying out deprotection by using 20% v/v piperidine/N, N-Dimethylformamide (DMF) solution of a deprotection reagent, carrying out deprotection reaction for 10min each time, repeating the deprotection reaction once, washing the resin by using 250ml of DMF, and washing for 6 times and 3min each time.
Fmoc-Tyr (tBu) -OH (20.67g,45.0mmol) and HOBt (6.69g,49.5mmol) were weighed into a conical flask, dissolved in 100.0ml of DMF, activated by adding DIC (7.76ml,49.5mmol) in an ice-water bath, and then added to a polypeptide synthesis reactor, reacted at room temperature for 2.0h, and the end of the reaction was checked by Kaiser test (negative colorless). Repeating the Fmoc-Tyr (tBu) -OH coupling steps (the feeding times are consistent with the consumption of the condensing agent), and sequentially coupling with the corresponding Fmoc protected amino acids of the bivalirudin sequence, wherein the corresponding protected amino acids are as follows: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-Gly-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH and Fmoc-D-Phe-OH, and the side chain total protection bivalidine peptide resin obtained after removing the Fmoc protection has the following structure:
H-D-Phe-Pro-Arg (Pbf) -Pro-Gly-Gly-Gly-Gly-Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu) -Ile-Pro-Glu (OtBu) -Tyr (tBu) -Leu-resin.
Example 3: synthesis of bivalirudin peptide resin with fully protected side chain
Weighing 25.0g (substitution degree of 0.6mmol/g) of Fmoc-Leu-Wang resin with the synthetic mole number of 15.0mmol, adding the Fmoc-Leu-Wang resin into a polypeptide synthesis reactor, adding 250.0ml of DMF to swell the resin for 30min, then pumping off the DMF, carrying out deprotection by using 20% v/v piperidine/N, N-Dimethylformamide (DMF) solution of a deprotection reagent, carrying out deprotection reaction for 10min each time, repeating the deprotection reaction once, washing the resin by using 250ml of DMF, and washing for 6 times and 3min each time.
Weighing Fmoc-Tyr (tBu) -OH (20.67g,45.0mmol) and HOBt (6.69g,49.5mmol) into a conical flask, adding 100.0ml of DMF for dissolving, adding DIC (7.76ml,49.5mmol) into an ice-water bath for activation, adding into a polypeptide synthesis reactor, reacting for 2.0h at room temperature, detecting the reaction endpoint (negative colorless) by Kaiser test, repeating the Fmoc-Tyr (tBu) -OH coupling steps (feeding times and the amount of a condensing agent are consistent), and sequentially coupling with Fmoc protected amino acids corresponding to the sequence of the Piroval sequence, wherein D-Phe at the 1 position adopts Boc-D-Phe-OH, and the rest corresponding protected amino acids are: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-Gly-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH and Boc-D-Phe-OH to obtain a side chain total protection ratio ivastatin peptide resin, which has the following structure:
Boc-D-Phe-Pro-Arg (Pbf) -Pro-Gly-Gly-Gly-Gly-Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu) -Ile-Pro-Glu (OtBu) -Tyr (tBu) -Leu-resin.
Example 4: synthesis of bivalirudin crude product
50g of bivalirudin peptide resin obtained in example 2 is weighed and placed in a 1000ml round bottom flask, and 300ml of lysis reagent (preferably trifluoroacetic acid/H) is added under ice bath2O/thioanisole/TIS 90.0/5/2.5/2.5 by volume), reaction for 3h, suction filtration, washing the resin with 16ml TFA 3 times, combining the filtrate and washings, pouring into 2.0L of cold methyl tert-ether, settling, suction filtration to obtain a solid, washing the methyl tert-ether 6 times, and vacuum drying to obtain 26.67g of crude peptide with purity 95.03% and yield 99.40%.
Example 5: synthesis of bivalirudin crude product
50g of bivalirudin peptide resin obtained in example 3 is weighed and placed in a 1000ml round bottom flask, and 300ml of lysis reagent (preferably trifluoroacetic acid/H) is added under ice bath2O/thioanisole/TIS 90.0/5/2.5/2.5, volume ratio), reaction for 3h, suction filtration, washing the resin with 15ml TFA 3 times, combining the filtrate and washings, pouring into 2.0L of cold methyl tert-ether, settling, suction filtration to obtain a solid, washing the methyl tert-ether 6 times, and vacuum drying to obtain 26.55g of crude peptide with purity 96.26% and yield 99.44%.
Example 6: bivalirudin crude peptide purification
Dissolving bivalirudin crude peptide in the embodiment 4 in a mixed solution of acetonitrile and water, carrying out suction filtration, carrying out sample loading and purification on a filtrate by adopting a C18 or C8 reversed-phase chromatographic column, carrying out gradient elution on a mobile phase by adopting 50-100 mmol/L ammonium acetate solution and B-phase acetonitrile, and carrying out one-step purification to obtain a pure qualified solution with the purity of more than 96%; continuing to perform secondary purification, and adopting trifluoroacetic acid as a mobile phase: acetonitrile (0.1-100:100-0.1, v/v), the detection wavelength is 220nm, sample peaks are collected and qualified samples are combined, desalting and freeze-drying are carried out, bivalirudin refined peptide is obtained, the yield of the refined peptide after purification is 59.06%, and the purity is 99.82%.
Example 7: bivalirudin crude peptide purification
Dissolving bivalirudin crude peptide in the embodiment 5 in a mixed solution of acetonitrile and water, carrying out suction filtration, carrying out sample loading and purification on a filtrate by adopting a C18 or C8 reversed-phase chromatographic column, carrying out gradient elution on a mobile phase by adopting 50-100 mmol/L ammonium acetate solution and B-phase acetonitrile, and carrying out one-step purification to obtain a pure qualified solution with the purity of more than 96%; continuing to perform secondary purification, and adopting trifluoroacetic acid as a mobile phase: acetonitrile (0.1-100:100-0.1, v/v), the detection wavelength is 220nm, the sample peaks are collected and qualified samples are merged, desalting and freeze-drying are carried out, the bivalirudin refined peptide is obtained, the yield of the refined peptide after purification is 59.13%, and the purity is 99.85%.
Claims (6)
1. A preparation method of bivalirudin is characterized by comprising the following specific steps:
(1) adopting Fmoc-Leu-Wang resin or Fmoc-Leu-CTC resin as a solid phase carrier, adding a deprotection agent, and removing an Fmoc protecting group on the resin;
(2) sequentially coupling the resin with the Fmoc protecting group removed in the step (1) with Fmoc-protected amino acids according to bivalirudin sequence in the presence of an activating agent: Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-Gly-Gly-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-D-Phe-OH or Boc-D-Phe-OH, obtaining a side chain protected bivalirudin peptide resin:
R1-D-Phe-Pro-Arg (Pbf) -X-Y-Asn (Trt) -Gly-Asp (OtBu) -Phe-Glu (OtBu) -Ile-Pro-Glu (OtBu) -Tyr (tBu) -Leu-resin
Wherein R is1Is Fmoc or Boc;
x is Pro-Gly-Gly;
y is Gly-Gly;
the Fmoc-Pro-Gly-Gly-OH synthesis steps are as follows: adopting a liquid phase synthesis method, taking H-Gly-Gly-OH, Fmoc-Pro-Osu and sodium carbonate as raw materials, taking 1, 4-dioxane and water as solvents, carrying out TLC detection reaction, adding ethyl acetate which is 5-8 times of the feeding times of Fmoc-Pro-Osu, extracting, concentrating, crystallizing, recrystallizing, filtering and drying, wherein the molar ratio of H-Gly-Gly-OH to sodium carbonate is 1: 0.8-1.0, and the molar ratio of Fmoc-Pro-OSu to diglycine is 1: 1.2-1.4;
(3) cracking the bivalirudin peptide resin with the side chain protected obtained in the step (2), and settling to obtain a bivalirudin crude product;
(4) purifying and freeze-drying the bivalirudin crude product in the step (3) to obtain a bivalirudin fine product, wherein the purification adopts a reversed phase C18 or C8 preparation column, and a mobile phase A phase: performing gradient elution on 50-100 mmol/L ammonium acetate solution and B-phase acetonitrile, and performing one-step purification to obtain a pure qualified solution with the purity of more than 96%; and (3) performing gradient elution on the first pure qualified liquid by adopting mobile phase A phase 0.01-0.05% TFA/water and B phase acetonitrile at the flow rate of 600ml/min to complete salt conversion and purification, obtaining two pure qualified liquids, and concentrating and freeze-drying the qualified liquids to obtain the bivalirudin refined peptide sample.
2. The method for preparing bivalirudin according to claim 1, wherein the substitution degree of the Fmoc-Leu-Wang resin or the Fmoc-Leu-CTC resin in the step (1) is in the range of 0.40 to 0.80 mmol/g; the deprotection agent is 20% v/v piperidine/N, N-Dimethylformamide (DMF) solution, and the dosage of the deprotection agent is 6-12 times of the mass of the resin.
3. The method for preparing bivalirudin according to claim 2, wherein the substitution degree of Fmoc-Leu-Wang resin or Fmoc-Leu-CTC resin is in the range of 0.6-0.7 mmol/g, and the dosage of deprotection reagent is 8-10 times of the mass of the resin.
4. The method for preparing bivalirudin according to claim 1, wherein the Fmoc-protected amino acid is fed in the step (2) by a factor of 2.0 to 3.0 times the synthesis scale; the activating agent is one of HOBT/DIC, HOAT/DIC, HBTU/HOBT/DIEA and HATU/HOAT/DIEA, and the dosage of the activating agent is 2.2-3.3 times of that of the resin; the coupling time is 1-5 h.
5. The bivalirudin preparation method according to claim 1, wherein in the step (3), the used amount of the lysis solution for the cleavage is 90-95% of TFA and one or more of thioanisole, dithioglycol, phenol, water and TIS, the amount of the lysis solution is 6-8 times of the mass of the peptide resin, and the cleavage reaction time is 2.0-3.0 h.
6. The method of claim 5, wherein the lysing solution is TFA/H2O/TIS/thioanisole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811101059.4A CN109134615B (en) | 2018-09-20 | 2018-09-20 | Preparation method of bivalirudin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811101059.4A CN109134615B (en) | 2018-09-20 | 2018-09-20 | Preparation method of bivalirudin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109134615A CN109134615A (en) | 2019-01-04 |
| CN109134615B true CN109134615B (en) | 2021-02-19 |
Family
ID=64815402
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811101059.4A Active CN109134615B (en) | 2018-09-20 | 2018-09-20 | Preparation method of bivalirudin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109134615B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110183532B (en) * | 2019-06-06 | 2023-06-30 | 济南爱思医药科技有限公司 | Process method for synthesizing bivalirudin protected pentapeptide fragments by mass high-efficiency liquid phase method |
| CN110204611B (en) * | 2019-06-26 | 2023-11-07 | 海南中和药业股份有限公司 | Solid phase fragment method for synthesizing bivalirudin |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010117725A2 (en) * | 2009-04-06 | 2010-10-14 | Novetide, Ltd. | Production of peptides containing poly-gly sequences using fmoc chemistry |
| CN102532274A (en) * | 2012-02-13 | 2012-07-04 | 成都圣诺生物制药有限公司 | Method for preparing bivalirudin |
| CN102924575A (en) * | 2012-10-31 | 2013-02-13 | 深圳翰宇药业股份有限公司 | Preparation method of bivalirudin |
| CN103242431A (en) * | 2013-05-20 | 2013-08-14 | 齐鲁制药有限公司 | Preparation method of bivalirudin |
| WO2014032257A1 (en) * | 2012-08-30 | 2014-03-06 | 深圳翰宇药业股份有限公司 | Method for preparing bivalirudin |
| CN104031127A (en) * | 2014-07-01 | 2014-09-10 | 济南康和医药科技有限公司 | Method for preparing bivalirudin through solid-liquid combination |
| CN104530224A (en) * | 2015-01-07 | 2015-04-22 | 哈尔滨吉象隆生物技术有限公司 | Bivalirudin solid-phase synthesis method |
| CN105218644A (en) * | 2015-11-09 | 2016-01-06 | 叶仲林 | A kind of preparation method of Bivalirudin |
| WO2017019174A1 (en) * | 2015-07-30 | 2017-02-02 | Ambiopharm, Inc. | Method of producing bivalirudin |
-
2018
- 2018-09-20 CN CN201811101059.4A patent/CN109134615B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010117725A2 (en) * | 2009-04-06 | 2010-10-14 | Novetide, Ltd. | Production of peptides containing poly-gly sequences using fmoc chemistry |
| CN102532274A (en) * | 2012-02-13 | 2012-07-04 | 成都圣诺生物制药有限公司 | Method for preparing bivalirudin |
| WO2014032257A1 (en) * | 2012-08-30 | 2014-03-06 | 深圳翰宇药业股份有限公司 | Method for preparing bivalirudin |
| CN102924575A (en) * | 2012-10-31 | 2013-02-13 | 深圳翰宇药业股份有限公司 | Preparation method of bivalirudin |
| CN103242431A (en) * | 2013-05-20 | 2013-08-14 | 齐鲁制药有限公司 | Preparation method of bivalirudin |
| CN104031127A (en) * | 2014-07-01 | 2014-09-10 | 济南康和医药科技有限公司 | Method for preparing bivalirudin through solid-liquid combination |
| CN104530224A (en) * | 2015-01-07 | 2015-04-22 | 哈尔滨吉象隆生物技术有限公司 | Bivalirudin solid-phase synthesis method |
| WO2017019174A1 (en) * | 2015-07-30 | 2017-02-02 | Ambiopharm, Inc. | Method of producing bivalirudin |
| CN105218644A (en) * | 2015-11-09 | 2016-01-06 | 叶仲林 | A kind of preparation method of Bivalirudin |
Non-Patent Citations (1)
| Title |
|---|
| "固相合成抗凝血药比伐卢定及合成条件的优化";沈忱等;《南京医科大学学报（自然科学版）》;20100930;第30卷(第9期);第1282-1286页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109134615A (en) | 2019-01-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106699871B (en) | Preparation method of liraglutide | |
| CN110294800B (en) | Preparation method of somaglutide | |
| CN101747426B (en) | Method for synthesizing pramlintide | |
| CN104788546A (en) | Preparation method of linear peptides containing 24 amino acid residues | |
| CN109134615B (en) | Preparation method of bivalirudin | |
| CN110698553A (en) | Preparation method of conus anti-wrinkle agent | |
| CN101104638A (en) | Solid phase synthetic technique for thymosin alpha1 | |
| CN101357938A (en) | Method for synthesizing Exenatide from solid phase polypeptide | |
| CN102229649B (en) | Preparation method of body protection polypeptide (BPC 157 peptide) | |
| CN106632655B (en) | Preparation method of exenatide and product thereof | |
| CN110204611B (en) | Solid phase fragment method for synthesizing bivalirudin | |
| CN103265620B (en) | Somatostatin and preparation method thereof | |
| CN113748125A (en) | Glucagon-like peptide-1 (GLP-1) receptor agonists and analogs thereof | |
| CN110903352A (en) | Preparation method of cetrorelix | |
| US20220033440A1 (en) | An improved process for the preparation of plecanatide | |
| CN112062835B (en) | Preparation method of bivalirudin | |
| CN104530224A (en) | Bivalirudin solid-phase synthesis method | |
| CN105111301B (en) | A kind of preparation method of salmon calcitonin | |
| CN108383896A (en) | A kind of method of segment method synthesis Goserelin | |
| CN112175067B (en) | Preparation method of teduglutide | |
| CN102875639A (en) | Solid-phase synthetic method of peptide and peptide synthesized by same | |
| CN114805480A (en) | Preparation method of octreotide | |
| CN111892650A (en) | Solid-phase synthesis method of liraglutide | |
| CN110981939A (en) | Preparation method of polycaprolactam | |
| US11419919B1 (en) | High-purity adrenocorticotropic hormone, analogue and a large-scale preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |